Efficient and reproducible differentiation protocol for pluripotent stem cells into functional endothelial cells: Unveiling the path to vascular regeneration

Introduction: Endothelial cells (ECs) play a crucial role in many treatments for cardiovascular diseases, such as blood vessel repair, tissue engineering, and drug delivery. The process of differentiating these cells is complex and involves various sources and numerous molecular and cellular events. Differentiating pluripotent stem cells (PSCs) into endothelial cells are one of the most effective sources for creating ECs in the lab and offers great potential for regenerative medicine. However, different cell types can appear during differentiation process. Objectives: This study presents a reliable and reproducible protocol for efficiently differentiating human pluripotent stem cells (hPSCs) into mature endothelial cells with high purity (>98%). Methods: FLK1+ cells were isolated from hPSCs using fluorescence-activated cell sorting (FACS). Then isolated FLK1+ cells differentiated into high-purity endothelial cells (ECs) by adding endothelial growth factors (VEGF, FGF, and EGM-2 medium). The differentiated ECs were extensively characterized by evaluating key endothelial markers and assessing their functional abilities, such as tube formation and response to angiogenic signals. Finally, the ECs were further purified using a second FACS step with a CD31 antibody. Results: The differentiated hPSC-derived endothelial cells (hPSC-ECs) expressed high levels of PECAM-1 (CD31), VE-cadherin (CD144), and von Willebrand factor (vWF), with more than 98% of the cells showing these markers. Additionally, the hPSC-ECs formed tubular structures and effectively took up acetylated fluorescently-labeled low-density lipoprotein (DiI-ac-LDL), demonstrating their functionality as endothelial cells. Conclusion: Our study clarifies the molecular mechanisms involved in the differentiation of hPSCs into endothelial cells, emphasizing key signaling pathways important for determining endothelial cell fate. These findings provide a framework for the scalable production of transplantable endothelial cells, representing a significant advancement in personalized therapies and tissue engineering for regenerative medicine.